Abstract: Amyloids play an integral role in a broad range of human illnesses including prion diseases, neurodegenerative conditions such as Alzheimer's and Parkinson's diseases, systemic amyloidoses, type II diabetes, and bacterial biofilms. Amyloids are aggregates of proteins or peptides with cross-beta structure and fibrillar morphology. Amyloids assemble via a rate-limiting nucleation step followed by fibril propagation in a dynamic environment with monomers, oligomers, and multimers. Effective therapeutics are urgently needed to treat amyloid-associated diseases but few options exist. For example, human prion diseases are rapidly fatal and untreatable. No disease-modifying therapies for Alzheimer's disease are available. No approved therapies directly treat biofilms, which serve to protect bacteria from antimicrobial agents. To create effective anti-amyloid therapeutics, the mechanisms that confer amyloidogenicity and toxicity must be better understood. Here, we propose to use synthetic biology and nanotechnology to understand and treat the pathogenic mechanisms underlying amyloid-associated diseases. Using peptide arrays, oligomer-binding antibodies, and a novel discovery platform for amyloid-toxicity inhibitors, we will identify peptides that modulate amyloid assembly and toxicity. These findings will be used to study the molecular mechanisms which underlie amyloid pathogenesis and to create effective multivalent peptide-displaying nanoparticles as therapeutic candidates. In particular, we shall: i) identify amyloid nucleation domains using peptide arrays and isolate oligomer-binding peptides, ii) produce multivalent peptide-nanoparticles that modulate amyloid assembly and oligomers, and iii) elucidate the conformational species and mechanisms which underlie amyloid toxicity using in vitro characterization, cell toxicity assays, and an innovative synthetic-biology-based platform for screening, selecting, and evolving toxicity inhibitors. This work will shed insights into the mechanisms that link amyloid assembly, oligomers, and toxicity using peptide and nanoparticle modulators. We will test the hypothesis that certain oligomers are pathogenic by altering assembly pathways and oligomer species with engineered peptide-nanoparticles. We shall also explore whether peptide-nanoparticles can decrease mammalian cell toxicity by directing assembly pathways towards non-toxic intermediates and fibrils. Identifying amyloid modulators will reveal the molecular pathways which govern the formation and pathogenesis of toxic species. We will also develop an innovative and broad platform to screen, select, and enrich for inhibitors of mammalian cell toxicity by amyloids. In addition, this system can be applied to discover peptide inhibitors of other diseases, such as viral infections and tauopathies. This high-throughput platform is enabled by our expertise in synthetic biology and bacterial engineering. Public Health Relevance: Amyloids are centrally involved in a broad range of important human illnesses, including prion diseases, neurodegenerative conditions such as Alzheimer's and Parkinson's diseases, systemic amyloidoses, type II diabetes, and bacterial biofilms but few amyloid treatments are available. This proposal aims to identify peptides which modulate amyloid assembly and toxicity using peptide arrays, oligomer-binding antibodies, and a novel discovery platform enabled by synthetic biology. The resulting findings will be used to study the molecular mechanisms which underlie amyloid toxicity and to create effective multivalent peptide-displaying nanoparticles as therapeutic candidates for amyloid-associated diseases.